ABSTRACT
Compensatory hepatocyte proliferation and other liver regenerative processes are activated to sustain normal physiological function after liver injury. A major mitogen for liver regeneration is hepatocyte growth factor (HGF), and a previous study indicated that progranulin could modulate c-met, the receptor for HGF, to initiate hepatic outgrowth from hepatoblasts during embryonic development. However, a role for progranulin in compensatory hepatocyte proliferation has not been shown previously. Therefore, this study was undertaken to clarify whether progranulin plays a regulatory role during liver regeneration. To this end, we established a partial hepatectomy regeneration model in adult zebrafish that express a liver-specific fluorescent reporter. Using this model, we found that loss of progranulin A (GrnA) function by intraperitoneal-injection of a Vivo-Morpholino impaired and delayed liver regeneration after partial hepatectomy. Furthermore, transcriptome analysis and confirmatory quantitative real-time PCR suggested that cell cycle progression and cell proliferation was not as active in the morphants as controls, which may have been the result of comparative downregulation of the HGF/c-met axis by 36 h after partial hepatectomy. Finally, liver-specific overexpression of GrnA in transgenic zebrafish caused more abundant cell proliferation after partial hepatectomy compared to wild types. Thus, we conclude that GrnA positively regulates HGF/c-met signaling to promote hepatocyte proliferation during liver regeneration.
Subject(s)
Hepatectomy/methods , Hepatocyte Growth Factor/metabolism , Hepatocytes/cytology , Liver Regeneration , Progranulins/metabolism , Proto-Oncogene Proteins c-met/metabolism , Zebrafish Proteins/metabolism , Animals , Cell Proliferation , Hepatocyte Growth Factor/genetics , Hepatocytes/metabolism , Organogenesis , Progranulins/genetics , Proto-Oncogene Proteins c-met/genetics , Signal Transduction , Zebrafish , Zebrafish Proteins/geneticsABSTRACT
BACKGROUND: Tilapia (Oreochromis niloticus) cultures are frequently infected by Vibrio vulnificus, causing major economic losses to production units. Previously, tilapia expressing recombinant delta-5 desaturase and delta-6 desaturase (D56) were found to be resistant to V. vulnificus infection. In this report, we profile the D56-mediated molecular changes underlying this resistance in tilapia. A comparative transcriptome analysis was performed on V. vulnificus-infected wild-type and D56-transgenic tilapia using Illumina's sequencing-by-synthesis approach. Gene enrichment analysis on differentially expressed unigenes was performed, and the expression patterns were validated by real-time PCR. RESULTS: Comparative transcriptome analysis was performed on RNA-sequence profiles obtained from wild-type and D56-transgenic tilapia at 0, 6 and 24 h post-infection with V. vulnificaus. GO and KEGG gene enrichment analyses showed that D56 regulates several pathways and genes, including fatty acid (FA) metabolism associated, and inflammatory and immune response. Expression of selected FA metabolism-associated, inflammatory and immune responsive genes was validated by qPCR. The inflammatory and immune responsive genes that are modulated by FA-associated D56 likely contribute to the enhanced resistance against V. vulnificus infection in Tilapia. CONCLUSIONS: Transcriptome profiling and filtering for two-fold change variation showed that 3795 genes were upregulated and 1839 genes were downregulated in D56-transgenic tilapia. These genes were grouped into pathways, such as FA metabolism, FA elongation, FA biosynthesis, biosynthesis of unsaturated FA, FA degradation, inflammation, immune response, and chemokines. FA-associated genes and immune-related genes were modulated by D56 at 6 h and 24 h post infection with V. vulnificus. The expression patterns of FA-related genes, inflammatory genes, antimicrobial peptide genes and immune responsive genes at 0, 3, 6, 12, 24 and 48 h post-infection suggests these genes are involved in the enhanced resistance of D56 transgenic tilapia to V. vulnificus.
Subject(s)
Cichlids , Fish Diseases , Tilapia , Vibrio Infections , Vibrio vulnificus , Animals , Cichlids/genetics , Fish Diseases/genetics , Gene Expression Profiling , Tilapia/genetics , Transcriptome , Vibrio Infections/genetics , Vibrio Infections/veterinary , Vibrio vulnificus/geneticsABSTRACT
Omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exhibit antibacterial and anti-inflammatory activities. Furthermore, diets rich in n-3 PUFAs are known to improve disease resistance and limit pathogen infection in commercial aquaculture fishes. In this study, we examined the effects of transgenic overexpression of n-3 PUFA biosynthesis genes on the physiological response to bacterial infection in tilapia. We first established tilapia strains with single or dual expression of salmon delta-5 desaturase and/or delta-6 desaturase and then challenged the fish with Vibrio vulnificus infection. Interestingly, our data suggest that n-3 PUFA-mediated alterations in gut microbiota may be important in determining disease outcome via effects on immune response of the host. Both liver- and muscle-specific single and dual expression of delta-5 desaturase and delta-6 desaturase resulted in higher n-3 PUFA content in transgenic fish fed with a LO basal diet. The enrichment of n-3 PUFAs in dual-transgenic fish is likely responsible for their improved survival rate and comparatively reduced expression of inflammation- and immune-associated genes after V. vulnificus infection. Gut microbiome analysis further revealed that dual-transgenic tilapia had high gut microbiota diversity, with low levels of inflammation-associated microbiota (i.e., Prevotellaceae). Thus, our findings indicate that dual expression of transgenic delta-5 and delta-6 desaturase in tilapia enhances disease resistance, an effect that is associated with increased levels of n-3 PUFAs and altered gut microbiota composition.
Subject(s)
Disease Resistance , Fatty Acid Desaturases/metabolism , Fish Proteins/metabolism , Gastrointestinal Microbiome , Linoleoyl-CoA Desaturase/metabolism , Tilapia/microbiology , Vibrio vulnificus/pathogenicity , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/microbiology , Delta-5 Fatty Acid Desaturase , Diet/veterinary , Discriminant Analysis , Disease Resistance/genetics , Docosahexaenoic Acids/metabolism , Fatty Acid Desaturases/genetics , Fatty Acids, Omega-3/metabolism , Fish Diseases/microbiology , Fish Diseases/pathology , Fish Proteins/genetics , Gene Expression , Least-Squares Analysis , Linoleoyl-CoA Desaturase/genetics , Tilapia/genetics , Vibrio Infections/pathology , Vibrio Infections/veterinaryABSTRACT
Cholangiocarcinoma (CCA) is a highly malignant cancer of the bile duct, which has a five-year survival rate less than 5% due to a high metastasis rate and lack of therapeutic options. Although omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to inhibit the proliferation of CCA cells, the effects on CCA metastasis have not been previously reported. In this study, we first assessed the proliferation, migration and invasion effects of n-3 PUFA-based fish oil on human CCA cells. Then, we investigated PUFA effects on metastasis in vivo by xenografting CCA cells into zebrafish larvae that overexpress a critical n-3 PUFA synthesis gene, Δ6 fatty acid desaturase. The results indicated that n-3 PUFA-based fish oil suppresses CCA cell growth, potentially by blocking the cell cycle at G2/M phase, and it inhibits migration and invasion potential with coincident downregulation of migration-related genes. Furthermore, zebrafish endogenous n-3 PUFAs appear to suppress CCA metastasis by inhibiting the expression of twist, a key regulator of tumor metastasis. Interestingly, only long chain n-3 PUFAs could inhibit the expression of twist in CCA cells. Together, our results suggest that n-3 PUFAs, especially DHA, may inhibit proliferation and metastasis of CCA cells by inhibiting the expression of twist.
Subject(s)
Bile Duct Neoplasms/diet therapy , Cholangiocarcinoma/diet therapy , Fatty Acids, Omega-3/pharmacology , Nuclear Proteins/genetics , Twist-Related Protein 1/genetics , Animals , Animals, Genetically Modified , Bile Duct Neoplasms/pathology , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Survival/drug effects , Cholangiocarcinoma/pathology , Fatty Acids, Omega-3/chemistry , Fish Oils/chemistry , Fish Oils/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Larva/drug effects , Xenograft Model Antitumor Assays , Zebrafish/geneticsABSTRACT
Liver cancer is the second leading cause of death worldwide. As such, establishing animal models of the disease is important for both basic and translational studies that move toward developing new therapies. Gankyrin is a critical oncoprotein in the genetic control of liver pathology. In order to evaluate the oncogenic role of gankyrin without cancer cell inoculation and drug treatment, we overexpressed gankyrin under the control of the fabp10a promoter. A Tet-Off system was used to drive expression in hepatocytes. At seven to twelve months of age, gankyrin transgenic fish spontaneously incurred persistent hepatocyte damage, steatosis, cholestasis, cholangitis, fibrosis and hepatic tumors. The tumors were both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). ICC is the second most frequent primary liver cancer in human patients and the first to develop in this tumor model. We further investigated the role of complement C3, a central molecule of the complement system, and found the expression levels of both in mRNA and protein are decreased during tumorigenesis. Together, these findings suggest that gankyrin can promote malignant transformation of liver cells in the context of persistent liver injury. This transformation may be related to compensatory proliferation and the inflammatory microenvironment. The observed decrease in complement C3 may allow transforming cells to escape coordinated induction of the immune response. Herein, we demonstrate an excellent zebrafish model for liver cancers that will be useful for studying the molecular mechanisms of tumorgenesis.
Subject(s)
Carcinoma, Hepatocellular/genetics , Cholangiocarcinoma/genetics , Liver Neoplasms/genetics , Liver/pathology , Proteasome Endopeptidase Complex/genetics , Proto-Oncogene Proteins/genetics , Up-Regulation , Zebrafish Proteins/genetics , Zebrafish/genetics , Animals , Apoptosis , Carcinoma, Hepatocellular/pathology , Cell Proliferation , Cholangiocarcinoma/pathology , Disease Models, Animal , Fatty Liver/genetics , Fatty Liver/pathology , Gene Expression Regulation, Neoplastic , Liver/metabolism , Liver Cirrhosis/genetics , Liver Cirrhosis/pathology , Liver Neoplasms/pathologyABSTRACT
BACKGROUND: Highly desaturated n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are synthesized by desaturases and elongase. They exert hepatoprotective effects to prevent alcoholic fatty liver syndrome or cholestatic liver injury. However, it is unclear how n-3 PUFAs improve immune function in liver. Vibrio vulnificus, a gram-negative bacterial pathogen, causes high mortality of aquaculture fishes upon infection. Humans can become infected with V. vulnificus through open wounds or by eating raw seafood, and such infections may result in systemic septicemia. Moreover, patients with liver diseases are vulnerable to infection, and are more likely than healthy persons to present with liver inflammation following infection. This study quantified n-3 PUFAs and their anti-bacterial effects in Fadsd6 and Elvol5a transgenic zebrafish. RESULTS: Two transgenic zebrafish strains with strong liver specific expression of Fadsd6 and Elvol5a (driven by the zebrafish Fabp10 promoter) were established using the Tol2 system. Synthesis of n-3 PUFAs in these strains were increased by 2.5-fold as compared to wild type (Wt) fish. The survival rate in 24 h following challenge with V. vulnificus was 20 % in Wt, but 70 % in the transgenic strains. In addition, the bacteria counts in transgenic fish strains were significantly decreased. The expression levels of pro-inflammatory genes, such as TNF-α, IL-1ß, and NF-κB, were suppressed between 9 and 12 h after challenge. This study confirms the anti-bacterial function of n-3 PUFAs in a transgenic zebrafish model. CONCLUSIONS: Fadsd6 and Elvol5a transgenic zebrafish are more resistant to V. vulnificus infection, and enhance survival by diminishing the attendant inflammatory response.
Subject(s)
Docosahexaenoic Acids/biosynthesis , Eicosapentaenoic Acid/biosynthesis , Fish Diseases/metabolism , Vibrio Infections/metabolism , Vibrio Infections/veterinary , Vibrio vulnificus , Zebrafish/metabolism , Animals , Animals, Genetically Modified , Docosahexaenoic Acids/genetics , Eicosapentaenoic Acid/genetics , Fish Diseases/genetics , Fish Diseases/microbiology , Vibrio Infections/genetics , Zebrafish/genetics , Zebrafish/microbiologyABSTRACT
BACKGROUND: Orange-spotted grouper (Epinephelus coioides) with protogynous hermaphroditic features are one of the most economically important aquaculture species in Taiwan. However, larvae stage grouper are susceptible to infection by the bacterial pathogen Vibrio alginolyticus. To better understand the molecular mechanisms of the immune response to V. alginolyticus in Epinephelus coioides larvae, we used high-throughput deep sequencing technology to study the effect of infection on gene expression. RESULTS: A total of 114,851,002 reads were assembled, consisting of 9,687,355,560 nucleotides; these were further assembled into 209,082 contigs with a mean length of 372 bp. Gene ontology (GO) analysis of the transcriptome revealed 12 cellular component subcategories, 16 molecular function subcategories, and 42 biological process subcategories (P value <0.05). A total of 32664 Epinephelus coioides genes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG); 1504 differentially expressed genes (DEGs) were subsequently identified, in 12 categories (P value <0.05). Vibrio infection affected the expression of genes involved in complementation, coagulation cascades, pathogen (Staphylococcus aureus) infection, phagosome activity, antigen processing, and the antigen presentation pathway. CONCLUSION: We conclude that the complement pathway of innate immunity and the hepicidin antimicrobial peptide may play important roles in the defense of Epinephelus coioides larvae against V. alginolyticus, and the immune response may activate at 4 h after bacterial infection. These results implicate the complement pathway signal pathway in immunity during V. alginolyticus infection at early developmental stages, enhancing our understanding of the mechanisms underlying the immune response to Vibrio infection in Epinephelus coioides.
Subject(s)
Complement Activation/genetics , Fish Diseases/genetics , Fish Diseases/microbiology , Immunity, Innate/genetics , Transcriptome , Vibrio Infections/veterinary , Vibrio alginolyticus/immunology , Animals , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/metabolism , Complement System Proteins/immunology , Computational Biology , Fish Diseases/immunology , Fish Diseases/metabolism , Gene Expression , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Metabolic Networks and Pathways , Molecular Sequence Annotation , Phagocytosis/genetics , Phagocytosis/immunology , Signal TransductionABSTRACT
UNLABELLED: The mechanisms that mediate the initiation and development of intrahepatic cholangiocarcinoma (ICC) associated with hepatitis B and C virus (HBV and HCV, respectively) infection remain largely unclear. In this study we conditionally coexpressed hepatitis B virus X (HBx) and hepatitis C virus core (HCP) proteins in zebrafish livers, which caused fibrosis and consequently contributed to ICC formation at the age of 3 months. Suppressing the transgene expression by doxycycline (Dox) treatment resulted in the loss of ICC formation. The biomarker networks of zebrafish ICC identified by transcriptome sequencing and analysis were also frequently involved in the development of human neoplasms. The profiles of potential biomarker genes of zebrafish ICC were similar to those of human cholangiocarcinoma. Our data also showed that the pSmad3L oncogenic pathway was activated in HBx and HCP-induced ICC and included phosphorylation of p38 mitogen-activated proteinbase (MAPK) and p44/42 mitogen-activated protein kinase (ERK1/2), indicating the association with transforming growth factor beta 1 (TGF-ß1) signaling pathway in ICC. Bile duct proliferation, fibrosis, and ICC were markedly reduced by knockdown of TGF-ß1 by in vivo morpholinos injections. CONCLUSION: These results reveal that TGF-ß1 plays an important role in HBx- and HCP-induced ICC development. This in vivo model is a potential approach to study the molecular events of fibrosis and ICC occurring in HBV and HCV infection.
Subject(s)
Bile Duct Neoplasms/genetics , Bile Ducts, Intrahepatic , Cholangiocarcinoma/genetics , Disease Models, Animal , Hepacivirus , Trans-Activators/genetics , Viral Core Proteins/genetics , Zebrafish , Animals , Animals, Genetically Modified , Anti-Bacterial Agents/pharmacology , Bile Duct Neoplasms/pathology , Cholangiocarcinoma/pathology , Connective Tissue Growth Factor/genetics , Cyclin D1/genetics , Doxycycline/pharmacology , Gene Expression/drug effects , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 3/genetics , Smad2 Protein/genetics , Smad3 Protein/genetics , Transforming Growth Factor beta1/genetics , Up-Regulation , Vascular Endothelial Growth Factor A/genetics , Viral Regulatory and Accessory Proteins , Zebrafish Proteins/genetics , p38 Mitogen-Activated Protein Kinases/geneticsABSTRACT
The unfolded protein response (UPR) is a conserved and adaptive cellular response to increase cell survival during ER stress. XBP-1 spliced form (XBP-1S) generated by IRE1 endoribonuclease is a key transcriptional regulator in UPR to activate genes involved in protein folding and degradation to restore ER function. Although Akt activation was suggested to be a pro-survival pathway activated during ER stress, the signal to trigger Akt is still not clear. In this study, we report IGF1 transcription and Akt phosphorylation are enhanced in XBP-1S stably overexpressed clone of zebrafish embryonic cell line (ZF4). In addition, zebrafish IGF1 intron1 with predicted UPRE (XBP-1S binding sites) and ERSE (ATF6/XBP-1S binding site) linked with basal promoter could be activated by XBP-1S, not by XBP-1 unspliced form (XBP-1U). Furthermore, we demonstrate that expression of endogenous IGF1 is transiently induced as XBP-1 splicing during ER stress in parallel to ER chaperone GRP78/Hspa5 and ER resided E3 ubiquitin ligase Synoviolin in ZF4 cells by quantitative PCR. Our results suggest zebrafish XBP-1S not only activates genes responsible for protein folding, transporting, glycosylation and ER associated degradation but also activates anti-apoptosis signal via IGF1/Akt pathway in unfolded protein response to cope with ER stress.